Abstract

Cardiovascular disease is the leading cause of morbidity and mortality in the Western world owing to the limited regenerative capacity of the mammalian cardiovascular system. In lieu of new muscle synthesis, the human heart replaces necrotic tissue with deposition of a noncontractile scar. By contrast, the adult zebrafish is endowed with a remarkable regenerative capacity, capable of de novo cardiomyocyte (CM) creation and scar tissue removal when challenged with an acute injury. In these studies, we examined the contributions of the dynamically regulated microRNA miR-101a during adult zebrafish heart regeneration. We demonstrate that miR-101a expression is rapidly depleted within 3 days post-amputation (dpa) but is highly upregulated by 7-14 dpa, before returning to uninjured levels at the completion of the regenerative process. Employing heat-inducible transgenic strains and antisense oligonucleotides, we demonstrate that decreases in miR-101a levels at the onset of cardiac injury enhanced CM proliferation. Interestingly, prolonged suppression of miR-101a activity stimulates new muscle synthesis but with defects in scar tissue clearance. Upregulation of miR-101a expression between 7 and 14 dpa is essential to stimulate removal of the scar. Through a series of studies, we identified the proto-oncogene fosab (cfos) as a potent miR-101a target gene, stimulator of CM proliferation, and inhibitor of scar tissue removal. Importantly, combinatorial depletion of fosab and miR-101a activity rescued defects in scar tissue clearance mediated by miR-101a inhibition alone. In summation, our studies indicate that the precise temporal modulation of the miR-101a/fosab genetic axis is crucial for coordinating CM proliferation and scar tissue removal during zebrafish heart regeneration.

Sustained suppression of miR-101a leads to increased scar tissue. (A-F) Wild-type and Tg(hs:miR-101a-sp) hearts were resectioned, heat treated daily and collected at 7, 14 and 30 dpa for histology. Hearts were sectioned at 10 µm and stained with Acid Fucshin Orange G (AFOG) to detect muscle (brown), collagen (blue) and fibrin (red). Arrowheads in F mark newly regenerated muscle; dashed lines indicate approximate resection injury plane. The penetrance of each phenotype is indicated (number showing the illustrated phenotype/total number of samples). (G) Scarring indices were established by quantifying the percentage of collagen and fibrin within the total injury area at the indicated time points using CellProfiler. Prolonged depletion of miR-101a led to greater scar tissue at 14 and 30 dpa compared with control. (H) Injury size was comparable between the groups as shown by quantification of total injury area. n=5-14; *P<0.001 (Student's t-test); error bars represent s.e.m.

Fig. 4.

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Upregulation of miR-101a between 7…

Fig. 4.

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Upregulation of miR-101a between 7 and 14 dpa is essential for scar tissue…

Fig. 4.

Upregulation of miR-101a between 7 and 14 dpa is essential for scar tissue removal. (A-I) Control and Tg(hs:miR-101a-sp) animals were subjected to ventricular resection and heat treated at defined intervals. Hearts were collected at 30 dpa and processed for AFOG staining to identify scar tissue. (A-C) Heat-treated induced depletion of miR-101a from 0 to 7 dpa did not alter scar tissue removal between control and Tg(hs:miR-101a-sp) hearts. (D-F) Suppression of miR-101a expression between 7 and 30 dpa resulted in increased scarring in Tg(hs:miR-101a-sp) hearts, compared with controls. (G-I) miR-101a suppression from 14 to 30 dpa, however, led to normal scar tissue clearance in control and Tg(hs:miR-101a-sp) hearts. Dashed lines indicate approximate resection injury plane. (J) Quantification of scarring indices demonstrates that increased miR-101a expression at 7-14 dpa is required for scar tissue clearance. ct, control. n=4-8; *P<0.01 (Student's t-test); NS, not significant; error bars represent s.e.m.

Long-term miR-101a depletion results in muscle regeneration and defects in scar tissue clearance. (A-F) Control, Tg(hs:miR-101a-sp) and Tg(hs:miR-133a1-pre) hearts were resectioned, heat treated daily for 30 dpa and extracted for histology. Hearts were cryosectioned at 10 µm and stained with either AFOG (A-C) or antibodies directed against Tropomyosin (D-F). Compared with heat-treated control animals, Tg(hs:miR-101a-sp) hearts reveal more scar tissue and less new muscle regeneration in the wounded apex. Tg(hs:miR-133a1-pre) hearts, which overexpress miR-133a1, show defects in both new muscle regeneration and scar tissue removal. Brackets represent approximate injury area; arrowheads in F mark gaps in myocardium and the lack of muscle regeneration. (G-I) Quantification of scarring indices, Tropomyosin expression and injury area were performed with CellProfiler. n=6-7; *P<0.05 (Student's t-test); error bars represent s.e.m.